Lack of association between measles virus vaccine and
autism with enteropathy: a case-control study: Appendix S1
Mady Hornig1§, Thomas Briese1, Timothy Buie2, Margaret L. Bauman3, Gregory Lauwers4,
Ulrike Siemetzki1, Kimberly Hummel5, Paul A. Rota5, William J. Bellini5, John J. O’Leary6,
Orla Sheils6, Errol Alden7, Larry Pickering8, W. Ian Lipkin1*
August 13, 2008
Contents
Page
S1
Development of assays for MV RNA
2
S2
Positive and negative controls for real-time PCR
3
S3
Assay calibration and procedures
4
S4
Sequencing methods
4
References
4
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Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY, USA
Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Boston, MA, USA
Department of Neurology, Harvard Medical School and Departments of Neurology and Pediatrics and
Learning and Developmental Disabilities Evaluation and Rehabilitation Services (LADDERS), Massachusetts
General Hospital, Boston, MA, USA
Department of Pathology of Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA
Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Department of Histopathology, Trinity College Dublin, Dublin, Ireland
American Academy of Pediatrics, Elk Grove Village, IL, USA
National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention,
Atlanta, GA, USA
* Corresponding author (email, wil2001@columbia.edu; phone +1 212 342 9033; fax +1 212 342 9044)
§ Co-corresponding author (email, mady.hornig@columbia.edu; phone +1 212 342 9036; fax +1 212 342 9044)
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S1
Development of assays for MV RNA
MeH and MeF primer pairs targeting the H and F gene of MeV, respectively, were designed
using Primer Express Software (PE Applied Biosystems) based on the following GenBank
sequence entries representing MV vaccine strains: AF266291, AF266290, AF266289,
AF266288, AF266287, AF266286, AB046218, AB032167, and AB016162. Testing by real
time RT-PCR demonstrated the capacity of the new primer/probe sets to detect vaccine
strain and wild type sequences associated with outbreaks of human disease (data not
shown). Alignment of primer/probe sets and wildtype sequences is shown in Figure S1A and
S1B (Real time RT-PCR primers for detection of MV F and H gene, respectively). Probes
were labeled with the reporter FAM (6-carboxyfluorescein) and quencher TAMRA (6carboxytetramethylrhodamine) (TIB MolBiol). Primer and probe sets used for detection of F
and H gene transcripts and -actin are listed in Table S1.
2
S2
Positive and negative controls for real-time PCR
To synthesize RNA standards for assay optimization, the plasmids MeF and MeH (pGEMTEasy, Invitrogen) were linearized with Sal I. After phenol-chloroform/ethanol extraction, 750
ng of DNA were in vitro transcribed with T7 polymerase (mMESSAGE mMACHINE® T7
ULTRA Kit, Ambion). Linearized plasmid DNA and synthetic RNA transcripts were used in
serial dilution with human placenta DNA (25 ng/l, Sigma) or yeast tRNA (50 ng/l, Sigma) as
diluent to generate standard curves. Assay performance characteristics were validated using
viral nucleic acid from three measles isolates: 1) vaccine strain virus (Moraten, a derivative of
the Edmonston seed B strain; 2) MVi/Beijing.CHN/94-1 (Chinese wild type isolate); and 3)
MVi/Reuler.LUX/17.96 (European wild type isolate). Standards were cloned for each of the
respective gene target regions of these MV isolates.
Among these different standards, a detection threshold of at least 5 molecules plasmid
standard per reaction was determined for the matching vaccine strain (Moraten); for the two
divergent wild type isolates, sensitivity was reduced by 1-2 orders of magnitude, depending
on the number of nucleotide mismatches in primers and probes (Figure S1A and Figure
S1B). Using synthetic RNA transcripts generated from the Moraten vaccine strain sequence,
a detection threshold of 50 molecules per RT-PCR reaction was determined.
3
S3
Assay calibration and procedures
All sites employed TaqMan Reverse Transcription Reagents (PE# N808-0234), TaqMan
Universal PCR Master Mix (PE# 4304447) and either an ABI 7700 or ABI 7900 thermal cycler
platform. Real-time PCR assay reagents (TaqMan Universal PCR Master Mix, primers,
probes) and positive and negative quality control standards (DNA plasmid and RNA transcript
standards; human placenta DNA, yeast tRNA, and water negative controls) were shipped to
all three laboratories from CU prior to shipment of blinded RNA study samples. Quality
control plates were run in each laboratory for calibration using synthetic cRNA standards prior
to shipment of study samples. Results of calibration assays were transmitted directly to the
Biostatistics Core and reviewed prior to shipment of coded study specimens to ensure
integrity of password-protected data transfer procedures to a secure website and calibration
of equipment and assay performance characteristics in each laboratory, including detection
thresholds. RNA (2 g total RNA/99 l reaction) was subjected to reverse transcription (RT)
with random hexamer priming. Five l of cDNA from each RT reaction were run in triplicate in
each of five real-time PCR assay plates (both primer/probe sets for F gene and H gene of
MV, and the housekeeping gene), along with a full set of positive and negative controls on
each plate. Thermocycling conditions for real-time PCR were: 2 min at 50C, 10 min at 95C
(polymerase activation), 45 cycles of 15 sec each at 95C (denaturation), and 1 min at 60C
(annealing, extension). Performance criteria were set to detection of 5-50 molecules and 50500 molecules in the DNA and RNA dilution series, respectively.
S4
Sequencing methods
Amplification products were size-fractionated in 1.3% agarose gels and visualized by
ethidium bromide staining at the end of the run. Products were sequenced after cloning into
pGEM-Teasy plasmid vectors (Promega). Sequences were obtained for both strands by
automated dideoxy-sequencing using BigDye Terminator Cycle Sequencing kits on ABI
Prism Genetic Analyzer systems (Applied Biosytems).
References
1.
2.
Uhlmann V, Martin CM, Sheils O, Pilkington L, Silva I, et al. (2002) Potential viral
pathogenic mechanism for new variant inflammatory bowel disease. Mol Pathol 55: 8490.
Kreuzer KA, Lass U, Landt O, Nitsche A, Laser J, et al. (1999) Highly sensitive and
specific fluorescence reverse transcription-PCR assay for the pseudogene-free
detection of beta-actin transcripts as quantitative reference. Clin Chem 45: 297-300.
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